目的:探讨大鼠慢性低灌注脑白质损伤中腺苷An 2A受体(An 2AR)的作用及其机制。n 方法:将180只成年雄性SD大鼠采用随机数字表法随机化分为假手术组(SG组，n n＝60)、慢性低灌注组(CG组，n n＝60)、慢性低灌注加An 2AR抑制剂组(IG组，n n＝30)和慢性低灌注加An 2AR激动剂组(AG组，n n＝30)4组，每组按取材时间不同随机分为2、4、8周3个亚组，其中SG和CG组每个亚组20只，IG组和AG组每个亚组10只。CG组、IG组、AG组通过双侧颈总动脉结扎法建立慢性低灌注模型，建模1 h后IG组和AG组分别用An 2AR抑制剂(2 mg/kg)和激动剂(0.5 mg/kg)腹腔注射，CG组和SG组予生理盐水(5 mL/kg)腹腔注射，每天1次，直到各组观察终点。4组大鼠分别在术后2、4、8周进行水迷宫实验，观察对比各组大鼠到达平台潜伏时间和穿越平台次数。水迷宫实验结束后，SG和CG组10只大鼠处死后取胼胝体，另外10只大鼠和IG、AG组10只大鼠取脑组织制备成冠状面切片。采用Kluver-Barrera染色观察4组大鼠脑组织的病理变化。SG和CG组大鼠采用Western blot检测胼胝体中An 2AR、DNA甲基转移酶(DNMTs)蛋白的表达水平，实时荧光定量聚合酶链反应(qPCR)检测胼胝体中An 2AR、DNMTs mRNA的表达水平，重亚硫酸盐测序法PCR(BSP)检测An 2AR基因启动子区域甲基化水平。n 结果:(1)水迷宫实验：SG组术后2、4、8周到达平台潜伏时间分别为(35.12±2.47)s、(37.64±1.59)s、(34.56±1.80)s，穿平台次数分别为(2.6±0.89)次、(2.6±0.54)次、(3.2±0.83)次；CG组术后2、4、8周到达平台潜伏时间分别为(39.58±0.82)s、(39.28±2.76)s、(42.44±2.84)s，穿平台次数分别为(1.4±0.54)次、(1.4±0.54)次、(1.6±0.55)次；IG组术后2、4、8周到达平台潜伏时间分别为(41.56±2.29)s、(44.26±1.60)s、(44.32±2.37)s，穿平台次数分别为(1.4±0.55)次、(1.0±0.71)次、(1.4±0.89)次；AG组术后2、4、8周到达平台潜伏时间分别为(37.64±2.41)s、(37.34±2.36)s、(39.24±1.73)s，穿平台次数分别为(1.8±0.45)次、(1.6±0.55)次、(1.8±0.83)次。SG、CG、IG组随低灌注时间延长到达平台潜伏时间呈上升趋势，差异均有统计学意义(n P值均0.05)。与SG组比较，CG组、IG组到达平台潜伏时间在术后2、4、8周时明显增加，AG组在术后2、8周时明显增加，差异均有统计学意义(n P值均<0.05)；与CG组比较，不同时间点IG组到达平台潜伏时间均明显增加，AG组均明显减少，差异均有统计学意义(n P值均<0.05)。SG组内随时间延长大鼠穿越平台次数呈上升趋势，差异有统计学意义(n P0.05)。与SG组比较，CG组、IG组、AG组不同时间点穿越平台次数均减少，差异有统计学意义(n P值均0.05)。(2)大鼠脑组织Kluver-Barrera染色结果显示：SG组大鼠神经纤维排列整齐，无空泡形成，神经纤维髓鞘完整。与SG组相比，CG组、IG组、AG组大鼠神经纤维变得疏松，部分纤维排列紊乱，局部出现空泡，且随着慢灌注时间的增加，变化更加明显。与CG组相比，IG组神经纤维变得更加疏松，纤维排列紊乱，空泡增加；AG神经纤维变得紧密，紊乱程度有所降低，空泡减少。(3)Western blot检测胼胝体An 2AR和DNMTs蛋白相对表达量：组内比较，CG组An 2AR、DNMT3a、DNMT3b蛋白的相对表达量随时间延长均呈下降趋势，差异均有统计学意义(n P值均0.05)。组间比较，CG组术后2、4、8周An 2AR蛋白的相对表达量均低于SG组，术后2、4、8周DNMT1、DNMT3a蛋白的相对表达量和术后2、4周DNMT3b蛋白的相对表达量均高于SG组，差异均有统计学意义(n P值均<0.05)。(4)qPCR检测胼胝体DNMTs mRNA表达水平：组内比较，CG组术后2、4、8周DNMT3a、DNMT3b mRNA的相对表达量随时间延长均呈先上升再下降趋势，差异有统计学意义(n P值均0.05)；而SG组DNMT1 mRNA随时间延长呈下降趋势(n P0.05)。组间比较，CG组术后2、4、8周An 2AR mRNA的相对表达量均低于SG组，DNMT1、DNMT3a、DNMT3b mRNA的相对表达量均高于SG组，差异均有统计学意义(n P值均<0.05)。(5)BSP检测甲基化水平：CG组在CpG12位点出现明显G锋，而SG组则转化为A锋。CG组甲基化位点比例15.83%(19/120)明显高于SG组的4.17%(5/120)，差异有统计学意义(χn 2＝9.074, n P<0.01)。n 结论:An 2AR在慢性低灌注脑白质损伤中发挥保护效应，其表达水平下调参与介导脑白质损伤，An 2AR表达下调可能与An 2AR DNA启动子区域甲基化水平增强有关。n “,”Objective:To investigate the effect and mechanism of adenosine An 2A receptor(An 2AR) in white matter lesions caused by chronic cerebral hypoperfusion.n Methods:A total of 180 adult male SD rats were divided into the sham operation group (SG group, n n=60) chronic hypoperfusion group (CG group, n n=60) chronic hypoperfusion plus An 2AR inhibitor group (IG group, n n=30), and chronic hypoperfusion plus An 2AR agonist group (AG group, n n=30) with the random number table method. According to sampling time, each group was randomly divided into three subgroups, namely, 2, 4, and 8 weeks groups. Each subgroup in the SG and CG groups had 20 rats each, whereas each subgroup in the IG and AG groups had 10 each. A cerebral chronic hypoperfusion model was set up with the bilateral common carotid arteries ligation method in the CG, IG, and AG groups. One hour after modeling, the IG and AG groups were intraperitoneally injected with An 2AR inhibitor (2 mg/kg) and agonist (0.5 mg/kg), respectively. The CG and SG groups were intraperitoneally injected with normal saline (5 mL/kg) once a day until the end of observation in each group. The four groups were subjected to water maze experiment 2, 4, and 8 weeks after operation, and the latency times of reaching the platform and times of crossing the platform were recorded and compared. After the water maze experiment, corpus callosum were collected from 10 rats in the SG and CG groups after death, and brain tissues were collected from the other 10 rats and 10 rats in IG and AG groups for the preparation of coronal plane sections. Kluver-Barrera staining was used in detecting pathological damage in the four groups. Western blot was used in detecting the An 2AR and DNA methyltransferase (DNMT) expression level in the cerebral white matter in the SG and CG groups. Quantitative real-time polymerase chain reaction (qPCR) was used in detecting DNMTs and An 2AR mRNA transcription levels in the SG and CG groups. The bisulfite sequence PCR(BSP) method was used in analyzing the A2A gene promoter region methylation level of the SG and CG groups.n Results:(1) Water maze: In the SG group, the latency of reaching the platform 2, 4, and 8 weeks after surgery was (35.12±2.47) s, (37.64±1.59) s, and (34.56±1.80)s, respectively, and the number of crossing the platform was (2.6±0.89), (2.6±0.54), and (3.2±0.83) times. In the CG group, the latency of reaching the platform 2, 4, and 8 weeks after surgery was (39.58±0.82) s, (39.28±2.76) s and (42.44±2.84)s and the number of crossing the platform was (1.4±0.54), (1.4±0.54), and (1.6±0.55) times, respectively. In the IG group, the latency of reaching the platform 2, 4, and 8 weeks after surgery was (41.56±2.29) s, (44.26±1.60) s, and (44.32±2.37)s, respectively, and the number of crossing the platform was (1.4±0.55), (1±0.71), and (1.4±0.89) times, respectively. In the AG group, the latency of reaching the platform 2, 4, and 8 weeks after operation was (37.64±2.41) s, (37.34±2.36) s, and (39.24±1.73)s, respectively, and the number of crossing the platform was (1.8±0.45), (1.6±0.55), and (1.8±0.83) times. The latency time to reach the platform in the SG, CG, and IG groups increased with time, and the differences were statistically significant (all n P values0.05). CG and IG groups were significantly increased 2, 4, and 8 weeks after surgery, compared with the SG group (alln P values<0.05), and the AG group showed significant increases 2 and 8 weeks after surgery, compared with the SG group (alln P values<0.05). The IG group showed significant increase at each time point, compared with the CG group (alln P values<0.05), and the AG group showed significant decrease at each time point, compared with the CG group (alln P values<0.05). The number of crossing the platform in SG group increased with time, and the difference was statistically significant (n P0.05). The CG, IG, and AG groups showed significant decreases at each time point, compared with the SG group (alln P values0.05). (2) Kluver-Barrera staining: the nerve fibers in the SG group were arranged neatly without vacuolation, and the myelin sheaths of the nerve fibers were complete. Compared with the nerve fibers in the SG group, the nerve fibers in the CG, IG, and AG groups became loose, and some fibers were disordered. Moreover, local vacuolation appeared. The changes became increasingly obvious over time. Compared with the nerve fibers in the CG group, the nerve fibers in the IG group became loose and disordered, and vacuolation increased. Compared with the nerve fibers of the CG group, the nerve fibers in the AG group became compact, the degree of disturbance was reduced, and vacuoles were reduced. (3) The relative protein expression levels of An 2AR and DNMTs in the corpus callosum were detected using Western blot. In the within-group comparison, the relative expression of An 2AR, DNMT3a, DNMT3b protein in the CG group decreased with time (all n P values0.05). In the comparison among groups, the expression of An 2AR protein in the CG group at 2, 4, and 8 weeks was lower than that in the SG group (all n P values<0.05), the expression of DNMT1 and DNMT3a protein at 2, 4, and 8 weeks and the relative expression of DNMT3b protein at 2 and 4 weeks were higher than that in the SG group (alln P values<0.05). (4) The mRNA expression level of DNMTs in the corpus callosum was detected with qPCR: In the within-group comparison, the mRNA relative expression levels of DNMT3a and DNMT3b in the CG group increased first and then decreased with time (alln P values0.05). The mRNA relative expression level of DNMT1 decreased in the SG group over time (n P0.05). In the comparison among groups, the mRNA relative expression levels of An 2AR in the CG group were lower than those in SG group 2, 4, and 8 weeks after surgery, and the mRNA relative expression levels of DNMT1, DNMT3a, and DNMT3b in the CG group were higher than those in the SG group 2, 4, and 8 weeks after surgery. The differences were statistically significant (all n P values<0.05). (5) Methylation level was measured with BSP: the CG group showed obvious G front at the CpG12 site, whereas the SG group changed to A front. The proportion of methylation sites in the CG group was 15.83% (19/120), which was significantly higher than that in the SG group (4.17%, 5/120). The difference was statistically significant (χn 2=9.074, n P<0.01).n Conclusions:An 2AR plays a protective role in chronic cerebral hypoperfusion white matter lesion. Its down-regulated expression level is involved in mediating cerebral white matter lesion. The down-regulated expression may be related to the enhanced methylation of An 2AR DNA promoter region.n